CN104418949A - Preparation method of teduglutide - Google Patents
Preparation method of teduglutide Download PDFInfo
- Publication number
- CN104418949A CN104418949A CN201310369843.4A CN201310369843A CN104418949A CN 104418949 A CN104418949 A CN 104418949A CN 201310369843 A CN201310369843 A CN 201310369843A CN 104418949 A CN104418949 A CN 104418949A
- Authority
- CN
- China
- Prior art keywords
- resin
- peptide
- otbu
- asp
- fmoc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the field of medicinal chemistry, and in particular relates to a preparation method of teduglutide. The preparation method of the invention is as below: respectively synthesizing a teduglutide sequence site No.4-33 peptide resin fragment and a teduglutide sequence No.1-3 peptide resin fragment through solid phase synthesis; then coupling the teduglutide sequence site No.4-33 peptide resin fragment and the teduglutide sequence No.1-3 peptide resin fragment by an appropriate resin vector to obtain a teduglutide resin; and finally cracking to obtain crude teduglutide, and purifying to obtain the pure teduglutide. The preparation method provided by the invention can effectively avoid side reaction such as degradation caused by an Asp-Gly structure of the No. 3-4 site, also can reduce the His1 racemization, thus effectively improving the purity of crude peptide and improve the purification yield. Compared with the prior art, the preparation method of the invention has the advanategs of high purity of the prepared teduglutide product, less by-product and high product yield, and is beneficial for mass production of teduglutide.
Description
Technical field
The invention belongs to medicinal chemistry art, particularly relate to a kind of preparation method for degree Shandong peptide.
Background technology
Short bowel syndrome (short bowel syndrome, SBS) refer to due to serious disease of intestine or surgical resection major part small intestine cause body cannot normal absorption nutrition and cause a series of syndrome.SBS reduces the ability that patient absorbs liquid and nutritive substance, and because remaining intestinal tube is too short, absorption of nutrient ingredients gastricism, can show as diarrhoea, steatorrhea, weight loss, and cause dehydration and malnutrition, severe patient is entail dangers to life even.Short bowel syndrome is many can not normal diet, and some is by recovering per os diet after actively appropriate Bowel nutritional rehabilitation, but some then needs to rely on parenteral nutrition throughout one's life.And Effect of Total Parenteral Nutrition will increase the risk of systemic infection and other long-term complications.There is no medicine up to now and can treat short bowel syndrome.
For degree Shandong peptide, English telaprevir by name, is the hormone of a kind of glucagon-like peptide 2 (GLP-2) analogue, a kind of natural generation, can reduces stomach emptying and secretion, and regulates the growth of small intestine endo cell, propagation and reparation.Orphan drug is named as in calendar year 2001 for degree Shandong peptide.Clinical trial shows, this medicine can reduce the demand of short bowel syndrome to parenteral alimentation.In June, 2012, European drug administration (EMA) approval is used for the treatment of adult's short bowel syndrome for degree Shandong peptide (teduglutide).Degree Shandong peptide molecule formula is replaced to be C
164h
252n
44o
55s, molecular weight is 3752.13, and amino acid is composed as follows: His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-lle-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-lle-Asn-Trp-Leu-lle-Gln-Thr-Lys-lle-Thr-Asp-OH.
At present about the preparation for degree Shandong peptide, publication number is that the method that have employed the coupling of pure solid phase order in the patent of CN101824087A carries out replacing the synthesis of spending Shandong peptide, concrete grammar is with wang resin for carrier, adopts conventional successive coupling, and final cutting obtains for degree Shandong peptide.And publication number is to solve the series of problems such as the degraded of the Asp-Gly fragment generation existed in the synthetic method of above-mentioned patent protection for degree Shandong peptide in WO2012028602A1 patent; adopt fragment coupling method to carry out and replace the synthesis of spending Shandong peptide; namely first full guard fragment 1-4 is synthesized; then the fragment 1-4 after adopting purifying and fragment peptide resin 5-33 carries out solid phase coupling, and final cracking obtains for degree Shandong peptide.
But researchist finds, no matter be adopt publication number to be the progressively coupling method of routine in the patent of CN101824087A or be the problem that WO2012028602A1 patent carries out that the preparation of solid phase fragment condensation approach all to exist the easy racemization of His1 for degree Shandong peptide according to publication number.Due to the racemization of His, cause the degree Shandong peptide that replaces obtained to be difficult to purifying, thus cause reducing for degree Shandong peptide yield.In addition, publication number is that the method that WO2012028602A1 patent adopts fragment 1-4 and fragment peptide resin 5-33 to carry out solid phase coupling is prepared for spending Shandong peptide, the side reactions such as the degraded that the Asp-Gly structure that still effectively can not solve 3-4 position is brought, the degree Shandong peptide that replaces obtained can be made equally to be difficult to purifying, to cause further reducing for degree Shandong peptide yield.
Summary of the invention
In view of this, the object of the invention is to the defect for prior art, provide the preparation method for degree Shandong peptide that a kind of yield is high, the side reactions such as the degraded that described preparation method can effectively avoid the Asp-Gly structure of 3-4 position to bring, also can reduce His simultaneously
1racemization, thus effectively improve thick peptide purity, improve purification yield.
For realizing object of the present invention, the present invention adopts following technical scheme:
For a preparation method for degree Shandong peptide, comprising:
Step 1: by Fmoc-Asp (OtBu)-OH and solid phase carrier coupling, obtain Fmoc-Asp (OtBu)-Resin;
Step 2:Fmoc-Asp (OtBu)-Resin is according to obtaining for degree Shandong peptide 4-33 fragment peptide resin H-Gly-Ser (tBu)-Phe-Ser (tBu)-Asp (OtBu)-Glu (OtBu)-Met-Asn (Trt)-Thr (tBu)-Ile-Leu-Asp (OtBu)-Asn (Trt)-Leu-Ala-Ala-Arg (pbf)-Asp (OtBu)-Phe-Ile-Asn (Trt)-Trp (Boc)-Leu-Ile-Gln (Trt)-Thr-Lys (Boc)-Ile-Thr (tBu)-Asp (OtBu)-Resin for the degree Shandong peptide order 4-32 amino acids for degree Shandong peptide that coupling Fmoc protects one by one;
Step 3: solid phase synthesis is for degree Shandong peptide 1-3 fragment peptide resin Boc-His (Trt)-Gly-Asp (OtBu)-Resin, and then cracking obtains for degree Shandong peptide full guard 1-3 peptide fragment Boc-His (Trt)-Gly-Asp (OtBu)-OH;
Step 4: for degree Shandong peptide full guard 1-3 peptide fragment, obtain for degree Shandong peptide resin for the coupling in solid phase of degree Shandong peptide 4-33 fragment peptide resin;
Step 5: obtain for the thick peptide of degree Shandong peptide for degree Shandong peptide resin cracking;
Step 6: obtain for degree Shandong peptide sterling for the thick peptide purification of degree Shandong peptide.
Preferably, solid phase carrier described in step 1 is selected from Wang Resin, PAM Resin, HMPA-AMResin, HMPA-MBHA Resin resin.
Preferably, the initial substitution degree of solid phase carrier described in step 1 is 0.5mmol/g ~ 1.0mmol/g.
Preferably, the substitution degree of Fmoc-Asp described in step 2 (OtBu)-Resin is 0.2 ~ 0.5mmol/g.
Preferably, the coupling reagent of coupling described in step 2 is HOBt/DIC.
Preferably, the carrier of solid phase synthesis described in step 3 is CTC resin.
Preferably, the cracking agent of cracking described in step 3 is the mixture of TFA and DCM or the mixture of TFE and DCM.
Preferably, the coupling reagent of coupling described in step 4 is HATU/HOAt/DIPEA.
Preferably, the cracking agent of cracking described in step 5 is the mixing solutions of TFA, PhSMe, EDT, TIS and water.
Preferably, purifying described in step 6 is RPLC purifying.
Preparation method of the present invention respectively solid phase synthesis spends peptide sequence 1-3 position, Shandong peptide resin fragment for spending Shandong peptide sequence 4-33 position peptide resin fragment and replacing, then with suitable resin for carrier, will for degree peptide sequence 4-33 position, Shandong peptide resin fragment and for spending the peptide resin fragment coupling of peptide sequence 1-3 position, Shandong, obtain for degree Shandong peptide resin, last cracking obtains for the thick peptide of degree Shandong peptide, and purifying obtains for degree Shandong peptide sterling.Of the present invention replacing is spent peptide sequence 4-33 position, Shandong peptide resin fragment and replaces the method spending peptide sequence 1-3 position, Shandong peptide resin fragment solid phase condensation, the side reactions such as the degraded can effectively avoiding the Asp-Gly structure of 3-4 position to bring, also can reduce His1 racemization simultaneously, thus effectively improve thick peptide purity, improve purification yield.In addition of the present invention for spending peptide sequence 4-33 position, Shandong peptide resin fragment and can carry out for the synthesis of spending peptide sequence 1-3 position, Shandong peptide resin fragment simultaneously, shorten synthesis cycle.Compared with prior art, preparation method of the present invention finally prepares for degree Shandong peptide product purity high, and by product is few, and product yield is high, is beneficial to the scale operation for degree Shandong peptide.
Embodiment
The embodiment of the invention discloses a kind of preparation method for degree Shandong peptide.Those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope method as herein described is changed or suitably change with combination, realize and apply the technology of the present invention.
For realizing object of the present invention, the present invention adopts following technical scheme:
For a preparation method for degree Shandong peptide, comprising:
Step 1: by Fmoc-Asp (OtBu)-OH and solid phase carrier coupling, obtain Fmoc-Asp (OtBu)-Resin;
Step 2:Fmoc-Asp (OtBu)-Resin is according to obtaining for degree Shandong peptide 4-33 fragment peptide resin H-Gly-Ser (tBu)-Phe-Ser (tBu)-Asp (OtBu)-Glu (OtBu)-Met-Asn (Trt)-Thr (tBu)-Ile-Leu-Asp (OtBu)-Asn (Trt)-Leu-Ala-Ala-Arg (pbf)-Asp (OtBu)-Phe-Ile-Asn (Trt)-Trp (Boc)-Leu-Ile-Gln (Trt)-Thr-Lys (Boc)-Ile-Thr (tBu)-Asp (OtBu)-Resin for the degree Shandong peptide order 4-32 amino acids for degree Shandong peptide that coupling Fmoc protects one by one;
Step 3:Thr-Lys (Boc)-Ile-Thr (tBu)-Asp (OtBu)-Resin;
Step 3: solid phase synthesis is for degree Shandong peptide 1-3 fragment peptide resin Boc-His (Trt)-Gly-Asp (OtBu)-Resin, and then cracking obtains for degree Shandong peptide full guard 1-3 peptide fragment Boc-His (Trt)-Gly-Asp (OtBu)-OH;
Step 4: for degree Shandong peptide full guard 1-3 peptide fragment, obtain for degree Shandong peptide resin for the coupling in solid phase of degree Shandong peptide 4-33 fragment peptide resin;
Step 5: obtain for the thick peptide of degree Shandong peptide for degree Shandong peptide resin cracking;
Step 6: obtain for degree Shandong peptide sterling for the thick peptide purification of degree Shandong peptide.
Preparation method for degree Shandong peptide of the present invention adopts Fmoc/tBu synthesis strategy, solid phase synthesis is for degree Shandong peptide sequence 4-33 position peptide resin fragment with for degree peptide sequence 1-3 position, Shandong peptide resin fragment respectively, then with suitable resin for carrier, will for degree peptide sequence 4-33 position, Shandong peptide resin fragment and for spending the peptide resin fragment coupling of peptide sequence 1-3 position, Shandong, obtain for degree Shandong peptide resin, last cracking obtains for the thick peptide of degree Shandong peptide, and purifying obtains for degree Shandong peptide.
First preparation method's step 1 of the present invention adopts Fmoc/tBu synthesis strategy, and Fmoc-Asp (OtBu)-OH is obtained Fmoc-Asp (OtBu)-Resin with solid phase carrier coupling.
Wherein, preferably, solid phase carrier described in step 1 is selected from Wang Resin, PAM Resin, HMPA-AM Resin, HMPA-MBHA Resin.Be more preferably Wang Resin.
Further, the initial substitution degree of solid phase carrier described in step 1 is preferably 0.5mmol/g ~ 1.0mmol/g.Be more preferably 1.0mmol/g
In some embodiments, preparation method's step 1 of the present invention be solid phase carrier and Fmoc-Asp (OtBu)-OH under the effect of coupling agent, be obtained by reacting Fmoc-Asp (OtBu)-Wang resin.Wherein, described coupling agent is preferably HOBt/DMAP/DIC.
Concrete, described Fmoc-Asp (OtBu)-OH first mixes with HOBt/DMAP, dissolves to add after DIC activates under ice bath in organic solvent to join in solid state reaction post, carries out linked reaction with the solid phase carrier of prior organic solvent dissolution.
As preferably, DCM and the DMF mixing solutions of the organic solvent of described dissolving Fmoc-Asp (OtBu)-OH to be volume ratio be 1:1.The organic solvent of the solid phase carrier of described dissolving is DMF.
As preferably, the condition of linked reaction is room temperature reaction 2 hours.
Linked reaction described in above-mentioned steps 1 needs after terminating to carry out purifying to reaction solution, is separated and obtains Fmoc-Asp (OtBu)-Resin.Described purification process concrete grammar is with DMF washing, adds confining liquid and closes, and then successively with DMF and DCM washing, methyl alcohol shrinks to be drained, and obtains Fmoc-Asp (OtBu)-Wang resin.Wherein, described confining liquid is preferably mol ratio is the pyridine of 1:1 and the mixing solutions of diacetyl oxide.Further, if if resin does not spread completely after closing, DCM need be added as dissolution with solvents.
Step 2 of the present invention obtains for degree Shandong peptide 4-33 fragment peptide resin H-Gly-Ser (tBu)-Phe-Ser (tBu)-Asp (OtBu)-Glu (OtBu)-Met-Asn (Trt)-Thr (tBu)-Ile-Leu-Asp (OtBu)-Asn (Trt)-Leu-Ala-Ala-Arg (pbf)-Asp (OtBu)-Phe-Ile-Asn (Trt)-Trp (Boc)-Leu-Ile-Gln (Trt)-Thr-Lys (Boc)-Ile-Thr (tBu)-Asp (OtBu)-Resin according to for the degree Shandong peptide order 4-32 amino acids for degree Shandong peptide that coupling Fmoc protects one by one on Fmoc-Asp (OtBu)-Resin.
Wherein, the substitution degree of Fmoc-Asp described in step 2 (OtBu)-Resin is 0.2mmol/g ~ 0.5mmol/g.
Further, the coupling reagent of coupling described in step 2 is HOBt/DIC.
In some embodiments, the method for the 4-32 amino acids spending Shandong peptide that coupling Fmoc protects one by one on Fmoc-Asp (OtBu)-Resin is specially: under the effect of the amino acid that Fmoc-Asp (OtBu)-Resin and Fmoc protect at coupling agent, coupling end reaction obtains for degree Shandong peptide 4-33 fragment peptide resin H-Gly-Ser (tBu)-Phe-Ser (tBu)-Asp (OtBu)-Glu (OtBu)-Met-Asn (Trt)-Thr (tBu)-Ile-Leu-Asp (OtBu)-Asn (Trt)-Leu-Ala-Ala-Arg (pbf)-Asp (OtBu)-Phe-Ile-Asn (Trt)-Trp (Boc)-Leu-Ile-Gln (Trt)-Thr-Lys (Boc)-Ile-Thr (tBu)-Asp (OtBu)-Resin one by one.Wherein, described coupling agent is preferably HOBt/DIC.
Concrete; amino acid Fmoc-Thr (the tBu)-OH of described Fmoc protection first mixes with HOBt; dissolve and join in solid state reaction post after adding DIC activation in organic solvent under ice bath, carry out linked reaction with Fmoc-Asp (the OtBu)-Resin of prior organic solvent dissolution.The 4-31 amino acids according to said method coupling one by one for degree Shandong peptide of Fmoc protection.
As preferably, DCM and the DMF mixing solutions of the amino acid whose organic solvent of described dissolving Fmoc protection to be volume ratio be 1:1.The organic solvent of Fmoc-Asp (the OtBu)-Resin of described dissolving is DMF.
As preferably, the condition of linked reaction described in above-mentioned steps 2 is room temperature reaction 2 hours.
Linked reaction described in above-mentioned steps 2 detects with ninhydrin method and judges each linked reaction terminal.If resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.
Linked reaction described in above-mentioned steps 2 needs after terminating to carry out purifying to reaction solution, is separated and obtains for degree Shandong peptide 4-33 fragment peptide resin.Described purification process concrete grammar is that linked reaction terminates the contraction of rear methyl alcohol, resin vacuum dried overnight, obtains for degree Shandong peptide 4-33 fragment peptide resin.
Step 3 solid phase synthesis of the present invention is for degree Shandong peptide 1-3 fragment peptide resin Boc-His (Trt)-Gly-Asp (OtBu)-Resin, and then cracking obtains for degree Shandong peptide full guard 1-3 peptide fragment Boc-His (Trt)-Gly-Asp (OtBu)-OH.
Wherein, solid phase synthesis described in step 3 is specially Fmoc-Asp (OtBu)-OH and carrier conjugation for degree Shandong peptide 1-3 fragment peptide resin Boc-His (Trt)-Gly-Asp (OtBu)-Resin and obtains Fmoc-Asp (OtBu)-Resin, then Fmoc-Asp (OtBu)-Resin one by one coupling Fmoc-Gly-OH and Boc-His (Trt)-OH obtain Boc-His (Trt)-Gly-Asp (OtBu)-Resin.
Preferably, the carrier of solid phase synthesis described in step 3 is CTC resin.The substitution degree of described CTC resin is preferably 1.0mmol/g.
In some embodiments, solid phase synthesis specifically comprises for the method for degree Shandong peptide 1-3 fragment peptide resin Boc-His (Trt)-Gly-Asp (OtBu)-Resin:
Step a, solid phase carrier CTC resin and Fmoc-Asp (OtBu)-OH linked reaction under the effect of coupling agent obtain Fmoc-Asp (OtBu)-CTC resin;
Obtain with Fmoc-Gly-OH and Boc-His (Trt)-OH linked reaction successively under the effect of step b, Fmoc-Asp (OtBu)-CTC resin coupling agents for spending Shandong peptide 1-3 fragment peptide resin Boc-His (Trt)-Gly-Asp (OtBu)-CTC resin.
Wherein, described in step a, coupling agent is preferably DMAP.Described in step b, coupling agent is preferably HOBt/DIC.
Concrete, linked reaction described in step a be specially Fmoc-Asp (OtBu)-OH dissolve add DMAP activation under ice bath in organic solvent after join in solid state reaction post, carry out linked reaction with the CTC resin of prior organic solvent dissolution.
As preferably, organic solvent described in step a is DMF.
As preferably, the condition of linked reaction described in above-mentioned steps a is again add DIPEA room temperature reaction 2 hours after room temperature reaction 5min.Adding DIPEA in batches, can to avoid just having started reaction too fierce, causes a large amount of heat release, ensure reaction temperature and.
Need after linked reaction described in above-mentioned steps a terminates to carry out purifying to reaction solution, be separated and obtain Fmoc-Asp (OtBu)-CTC resin.Described purification process concrete grammar is that linked reaction terminates rear DMF and washs, and adds confining liquid and closes, and then with DMF washing, methyl alcohol shrinks to be drained, and obtains Fmoc-Asp (OtBu)-CTC resin.Wherein, described confining liquid is preferably methyl alcohol.Further, if if resin does not spread completely after closing, DMF need be added as dissolution with solvents.
Further, linked reaction described in step b is specially described Fmoc-Gly-OH and first mixes with HOBt, dissolve and join in solid state reaction post after adding DIC activation in organic solvent under ice bath, carry out linked reaction with Fmoc-Asp (the OtBu)-CTC resin of prior organic solvent dissolution and obtain Fmoc-Gly-Asp (OtBu)-CTC resin.Boc-His (Trt)-OH according to said method obtains with the coupling of Fmoc-Gly-Asp (OtBu)-CTC resin for spending Shandong peptide 1-3 fragment peptide resin Boc-His (Trt)-Gly-Asp (OtBu)-CTC resin.
As preferably, organic solvent described in step b is DMF.
As preferably, the condition of linked reaction described in above-mentioned steps b is room temperature reaction 2 hours.
Linked reaction described in above-mentioned steps b detects with ninhydrin method and judges each linked reaction terminal.If resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.
Need after linked reaction described in above-mentioned steps b terminates to carry out purifying to reaction solution, be separated and obtain Boc-His (Trt)-Gly-Asp (OtBu)-CTC resin.Described purification process concrete grammar is that linked reaction terminates the contraction of rear methyl alcohol, resin vacuum dried overnight, obtains for degree Shandong peptide 1-3 fragment peptide resin Boc-His (Trt)-Gly-Asp (OtBu)-CTC resin.
Obtaining for degree Shandong peptide full guard 1-3 peptide fragment Boc-His (Trt)-Gly-Asp (OtBu)-OH for degree Shandong peptide 1-3 fragment peptide resin Boc-His (Trt)-Gly-Asp (OtBu)-Resin cracking of step 3 solid phase synthesis of the present invention.
Wherein, described in step 3, the cracking agent of cracking is preferably the mixture of TFA and DCM.
Further, in the mixture of TFA and DCM described in above-mentioned cracking agent, the volume ratio of TFA and DCM is preferably 0.5:99.5 ~ 5:95.In some embodiments, the volume ratio of TFA and DCM is 1:99.
Further, the add-on of cracking agent described in step 3 is preferably pressed mL/g and is calculated, and the ratio of cracking agent and peptide resin is 10:1.Namely every 1g peptide resin adds the above-mentioned cracking agent of 10mL.
Further, described in step 3, crack reacting condition is preferably stirring at room temperature 2.5h.
In some embodiments, scission reaction described in above-mentioned steps 3 needs after terminating to carry out purifying to reaction solution, is separated and obtains for degree Shandong peptide full guard 1-3 peptide fragment Boc-His (Trt)-Gly-Asp (OtBu)-OH.Described purification process concrete grammar is that reaction solution sand core funnel filters, collect filtrate, resin washs 3 times with a small amount of DCM again, concentrating under reduced pressure after merging filtrate, add freezing anhydrous diethyl ether precipitation, collecting precipitation anhydrous diethyl ether washs 3 times, and vacuum-drying obtains white powder solid, i.e. thick peptide Boc-His (Trt)-Gly-Asp (OtBu)-OH.Wherein DCM washing can wash out, the product be adsorbed on resin of remnants to reduce the loss as far as possible.
Preparation method's step 4 of the present invention obtains for degree Shandong peptide resin for degree Shandong peptide full guard 1-3 peptide fragment for the coupling in solid phase of degree Shandong peptide 4-33 fragment peptide resin.
Wherein, described in step 4, the coupling reagent of coupling is preferably HATU/HOAt/DIPEA.
In a specific embodiment; be specially for the method for degree Shandong peptide full guard 1-3 peptide fragment for the coupling in solid phase of degree Shandong peptide 4-33 fragment peptide resin described in step 4: first dissolve with HATU and HOAt to add after DIPEA activate under ice bath in organic solvent for degree Shandong peptide full guard 1-3 peptide fragment and join in solid state reaction post, spend Shandong peptide 4-33 fragment peptide resin and carry out linked reaction with replacing of prior organic solvent dissolution and obtain replacing and spend Shandong peptide peptide resin.
As preferably, organic solvent described in step 4 is DMF.
As preferably, the condition of linked reaction described in step 4 is room temperature reaction 2 hours.
Linked reaction described in above-mentioned steps 4 detects with ninhydrin method and judges linked reaction terminal.If resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again.
Linked reaction described in above-mentioned steps 4 needs after terminating to carry out purifying to reaction solution, is separated and obtains for degree Shandong peptide peptide resin.Described purification process concrete grammar is that linked reaction terminates the contraction of rear methyl alcohol, resin vacuum dried overnight, obtains for degree Shandong peptide peptide resin.
Preparation method's step 5 of the present invention will obtain for the thick peptide of degree Shandong peptide for degree Shandong peptide resin cracking.
Wherein, cracking agent described in step 5 is preferably the mixing solutions of TFA, PhSMe, EDT, TIS and water.Be more preferably TFA, PhSMe, EDT, TIS and water volume ratio is the mixing solutions of 86:5:5:3:1.
Further, the add-on of cracking agent described in step 5 is preferably pressed mL/g and is calculated, and the ratio of cracking agent and peptide resin is 10:1.Namely every 1g peptide resin adds the above-mentioned cracking agent of 10mL.
Further, described in step 5, crack reacting condition is preferably stirring at room temperature 2.5h.
In some embodiments, scission reaction described in above-mentioned steps 3 needs after terminating to carry out purifying to reaction solution, is separated and obtains for the thick peptide of degree Shandong peptide.Described purification process concrete grammar is that reaction solution sand core funnel filters, and collect filtrate, resin washs with a small amount of TFA again, concentrating under reduced pressure after merging filtrate, add freezing anhydrous diethyl ether precipitation, collecting precipitation anhydrous diethyl ether washs 3 times, and vacuum-drying obtains for the thick peptide of degree Shandong peptide.Wherein TFA washing can wash out, the product be adsorbed on resin of remnants to reduce the loss as far as possible.
Being further purified for the thick peptide of degree Shandong peptide that cracking obtains by preparation method's step 6 of the present invention obtains for degree Shandong peptide sterling, and wherein said purifying is preferably RPLC purifying.
RPLC, English name reversed phase high performance liquidchromatography, is called for short, RP-HPLC, the liquid chromatographic system be made up of non-polar stationary phase and polarity moving phase.It is just in time contrary with the liquid chromatographic system (normal-phase chromatography) be made up of polar stationary phase and low-pole moving phase.RP-HPLC is the topmost clastotype of current liquid chromatography, almost can be used for all organic separation and purification that can be dissolved in polarity or weak polar solvent.What step 5 cracking obtained by preparation method of the present invention adopts reversed-phased high performace liquid chromatographic purifying to obtain for degree Shandong peptide sterling for the thick peptide of degree Shandong peptide.
Preferably, described reversed-phased high performace liquid chromatographic is specially: with anti-phase octadecylsilane or eight alkyl silane bonded silica gels for stationary phase, after the Pa Xirui peptide crude product solution loading that cracking obtains, purify with 0.2%TFA/ acetonitrile mobile phase, collects object peak cut; With anti-phase octadecylsilane or eight alkyl silane bonded silica gels for stationary phase, turn salt with 0.1% acetum/acetonitrile mobile phase, namely freeze-drying obtains for degree Shandong peptide acetate essence peptide namely for degree Shandong peptide sterling.
In order to understand the present invention further, below in conjunction with embodiment, the present invention is described in detail.
The implication of the abbreviation used in specification sheets and claims is listed in the following table:
| Abbreviation and English | Implication |
| Wang Resin | King's resin |
| PAM Resin | 4-methylol-phenylacetamidomethyl resin |
| CTC Resin | 2-chlorine trityl chloride resin |
| HOAt | 1-hydroxyl-7-azo benzotriazole |
| Fmoc | 9-fluorenylmethyloxycarbonyl |
| HOBt | I-hydroxybenzotriazole |
| HATU | 2-(7-azo benzotriazole)-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester |
| HOAt | 1-hydroxyl-7-azo benzotriazole |
| DIPEA | DIPEA |
| Hmb | 2-hydroxyl-4-methoxy-benzyl |
| Dmb | 2,4-dimethoxy-benzyl |
| DMF | DMF |
| DCM | Methylene dichloride |
| TFE | Trifluoroethanol |
| TFA | Trifluoroacetic acid |
| PhSMe | PhSMe |
| TIS | Tri isopropyl silane |
| DIC | N, N'-DIC |
| EDT | Dithioglycol |
Embodiment 1: substitution degree is the synthesis of Fmoc-Asp (the OtBu)-Wang resin of 0.5mmol/g
Take the Wang resin 40g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 24.72g Fmoc-Asp (OtBu)-OH(60mmol), 9.73g HOBt(72mmol) and 0.73g DMAP(6mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 11.26mL DIC(72mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Wash 3 times with DMF, add 70mL confining liquid (pyridine/diacetyl oxide=1:1,400mmol:400mmol) and close 8 hours (if resin does not spread completely add DCM as solvent).Wash 4 times with DMF, DCM washes 4 times, and methyl alcohol shrinks to be drained, and obtains Fmoc-Asp (OtBu)-Wang resin.Detection substitution degree is 0.503mmol/g.
Embodiment 2: substitution degree is the synthesis of Fmoc-Asp (the OtBu)-Wang resin of 0.2mmol/g
Take the Wang resin 40g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 9.89g Fmoc-Asp (OtBu)-OH(24mmol), 3.90g HOBt(28.8mmol) and 0.29g DMAP(0.24mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 4.50mL DIC(28.8mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Wash 3 times with DMF, add 70mL confining liquid (pyridine/diacetyl oxide=1:1,400mmol:400mmol) and close 8 hours (if resin does not spread completely add DCM as solvent).Wash 4 times with DMF, DCM washes 4 times, and methyl alcohol shrinks to be drained, and obtains Fmoc-Asp (OtBu)-Wang resin.Detection substitution degree is 0.198mmol/g.
Embodiment 3: substitution degree is the synthesis of Fmoc-Asp (the OtBu)-Wang resin of 0.3mmol/g
Take the Wang resin 40g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 14.83g Fmoc-Asp (OtBu)-OH(36mmol), 5.84g HOBt(43.2mmol) and 1.44g DMAP(3.6mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 6.75mL DIC(43.2mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Wash 3 times with DMF, add 70mL confining liquid (pyridine/diacetyl oxide=1:1,400mmol:400mmol) and close 8 hours (if resin does not spread completely add DCM as solvent).Wash 4 times with DMF, DCM washes 4 times, and methyl alcohol shrinks to be drained, and obtains Fmoc-Asp (OtBu)-Wang resin.Detection substitution degree is 0.302mmol/g.
Embodiment 4:H-Gly-Ser, (tBu)-Phe-Ser, (tBu)-Asp, (OtBu)-Glu, (OtBu)-Met-Asn, (Trt)-Thr, (tBu)-Ile-Leu-Asp, (OtBu)-Asn, (Trt)-Leu-Ala-Ala-Arg, (pbf)-Asp, (OtBu)-Phe-Ile-Asn, (Trt)-Trp, (Boc)-Leu-Ile-Gln, (Trt)-Thr-Lys, (Boc)-Ile-Thr, (tBu)-Asp, (OtBu) preparation of-Wang Resin peptide resin
Take Fmoc-Asp (the OtBu)-Wang resin 33.1g that substitution degree in embodiment 3 is 0.302mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Get 11.93g Fmoc-Thr (tBu)-OH(30mmol), 4.86g HOBt(36mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, under ice-water bath, add 5.63mL DIC(36mmol) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to replacing degree Shandong peptide main chain peptide sequence, hold N to hold the coupling completing 4-33 fragment successively from C, reaction terminates rear methyl alcohol and shrinks; resin vacuum dried overnight, weighs and obtains for degree Shandong peptide (4-33)-Wang resin 76.0g.
Embodiment 5: substitution degree is the synthesis of Fmoc-Asp (the OtBu)-PAM resin of 0.5mmol/g
Take the PAM resin 40g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 24.72g Fmoc-Asp (OtBu)-OH(60mmol), 9.73g HOBt(72mmol) and 0.73g DMAP(6mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 11.26mL DIC(72mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Wash 3 times with DMF, add 70mL confining liquid (pyridine/diacetyl oxide=1:1,400mmol:400mmol) and close 8 hours (if resin does not spread completely add DCM as solvent).Wash 4 times with DMF, DCM washes 4 times, and methyl alcohol shrinks to be drained, and obtains Fmoc-Asp (OtBu)-PAM resin.Detection substitution degree is 0.516mmol/g.
Embodiment 6: substitution degree is the synthesis of Fmoc-Asp (the OtBu)-PAM resin of 0.2mmol/g
Take the PAM resin 40g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 9.89g Fmoc-Asp (OtBu)-OH(24mmol), 3.90g HOBt(28.8mmol) and 0.29g DMAP(0.24mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 4.50mL DIC(28.8mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Wash 3 times with DMF, add 70mL confining liquid (pyridine/diacetyl oxide=1:1,400mmol:400mmol) and close 8 hours (if resin does not spread completely add DCM as solvent).Wash 4 times with DMF, DCM washes 4 times, and methyl alcohol shrinks to be drained, and obtains Fmoc-Asp (OtBu)-PAM resin.Detection substitution degree is 0.189mmol/g.
Embodiment 7: substitution degree is the synthesis of Fmoc-Asp (the OtBu)-PAM resin of 0.3mmol/g
Take the PAM resin 40g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 14.83g Fmoc-Asp (OtBu)-OH(36mmol), 5.84g HOBt(43.2mmol) and 1.44g DMAP(3.6mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 6.75mL DIC(43.2mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Wash 3 times with DMF, add 70mL confining liquid (pyridine/diacetyl oxide=1:1,400mmol:400mmol) and close 8 hours (if resin does not spread completely add DCM as solvent).Wash 4 times with DMF, DCM washes 4 times, and methyl alcohol shrinks to be drained, and obtains Fmoc-Asp (OtBu)-PAM resin.Detection substitution degree is 0.298mmol/g.
Embodiment 8: substitution degree is the synthesis of Fmoc-Asp (the OtBu)-HMPA-AM resin of 0.5mmol/g
Take the HMPA-AM resin 40g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 24.72g Fmoc-Asp (OtBu)-OH(60mmol), 9.73g HOBt(72mmol) and 0.73g DMAP(6mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 11.26mL DIC(72mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Wash 3 times with DMF, add 70mL confining liquid (pyridine/diacetyl oxide=1:1,400mmol:400mmol) and close 8 hours (if resin does not spread completely add DCM as solvent).Wash 4 times with DMF, DCM washes 4 times, and methyl alcohol shrinks to be drained, and obtains Fmoc-Asp (OtBu)-HMPA-AM resin.Detection substitution degree is 0.503mmol/g.
Embodiment 9: substitution degree is the synthesis of Fmoc-Asp (the OtBu)-HMPA-AM resin of 0.2mmol/g
Take the HMPA-AM resin 40g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 9.89g Fmoc-Asp (OtBu)-OH(24mmol), 3.90g HOBt(28.8mmol) and 0.29g DMAP(0.24mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 4.50mL DIC(28.8mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Wash 3 times with DMF, add 70mL confining liquid (pyridine/diacetyl oxide=1:1,400mmol:400mmol) and close 8 hours (if resin does not spread completely add DCM as solvent).Wash 4 times with DMF, DCM washes 4 times, and methyl alcohol shrinks to be drained, and obtains Fmoc-Asp (OtBu)-HMPA-AM resin.Detection substitution degree is 0.214mmol/g.
Embodiment 10: substitution degree is the synthesis of Fmoc-Asp (the OtBu)-HMPA-AM resin of 0.3mmol/g
Take the HMPA-AM resin 40g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 14.83g Fmoc-Asp (OtBu)-OH(36mmol), 5.84g HOBt(43.2mmol) and 1.44g DMAP(3.6mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 6.75mL DIC(43.2mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Wash 3 times with DMF, add 70mL confining liquid (pyridine/diacetyl oxide=1:1,400mmol:400mmol) and close 8 hours (if resin does not spread completely add DCM as solvent).Wash 4 times with DMF, DCM washes 4 times, and methyl alcohol shrinks to be drained, and obtains Fmoc-Asp (OtBu)-HMPA-AM resin.Detection substitution degree is 0.309mmol/g.
Embodiment 11: substitution degree is the synthesis of Fmoc-Asp (the OtBu)-HMPA-MBHA resin of 0.5mmol/g
Take the HMPA-AM resin 40g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 24.72g Fmoc-Asp (OtBu)-OH(60mmol), 9.73g HOBt(72mmol) and 0.73g DMAP(6mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 11.26mL DIC(72mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Wash 3 times with DMF, add 70mL confining liquid (pyridine/diacetyl oxide=1:1,400mmol:400mmol) and close 8 hours (if resin does not spread completely add DCM as solvent).Wash 4 times with DMF, DCM washes 4 times, and methyl alcohol shrinks to be drained, and obtains Fmoc-Asp (OtBu)-HMPA-MBHA resin.Detection substitution degree is 0.503mmol/g.
Embodiment 12: substitution degree is the synthesis of Fmoc-Asp (the OtBu)-HMPA-MBHA resin of 0.2mmol/g
Take the HMPA-MBHA resin 40g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 9.89g Fmoc-Asp (OtBu)-OH(24mmol), 3.90g HOBt(28.8mmol) and 0.29g DMAP(0.24mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 4.50mL DIC(28.8mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Wash 3 times with DMF, add 70mL confining liquid (pyridine/diacetyl oxide=1:1,400mmol:400mmol) and close 8 hours (if resin does not spread completely add DCM as solvent).Wash 4 times with DMF, DCM washes 4 times, and methyl alcohol shrinks to be drained, and obtains Fmoc-Asp (OtBu)-HMPA-MBHA resin.Detection substitution degree is 0.203mmol/g.
Embodiment 13: substitution degree is the synthesis of Fmoc-Asp (the OtBu)-HMPA-MBHA resin of 0.3mmol/g
Take the HMPA-MBHA resin 40g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 14.83g Fmoc-Asp (OtBu)-OH(36mmol), 5.84g HOBt(43.2mmol) and 1.44g DMAP(3.6mmol) be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, add 6.75mL DIC(43.2mmol under ice-water bath) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Wash 3 times with DMF, add 70mL confining liquid (pyridine/diacetyl oxide=1:1,400mmol:400mmol) and close 8 hours (if resin does not spread completely add DCM as solvent).Wash 4 times with DMF, DCM washes 4 times, and methyl alcohol shrinks to be drained, and obtains Fmoc-Asp (OtBu)-HMPA-MBHA resin.Detection substitution degree is 0.310mmol/g.
Embodiment 14:H-Gly-Ser, (tBu)-Phe-Ser, (tBu)-Asp, (OtBu)-Glu, (OtBu)-Met-Asn, (Trt)-Thr, (tBu)-Ile-Leu-Asp, (OtBu)-Asn, (Trt)-Leu-Ala-Ala-Arg, (pbf)-Asp, (OtBu)-Phe-Ile-Asn, (Trt)-Trp, (Boc)-Leu-Ile-Gln, (Trt)-Thr-Lys, (Boc)-Ile-Thr, (tBu)-Asp, (OtBu) preparation of-Pam Resin peptide resin
Take Fmoc-Asp (the OtBu)-PAM resin 33.6g that substitution degree in embodiment 7 is 0.298mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Get 11.93g Fmoc-Thr (tBu)-OH(30mmol), 4.86g HOBt(36mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, under ice-water bath, add 5.63mL DIC(36mmol) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to replacing degree Shandong peptide main chain peptide sequence, hold N to hold the coupling completing 4-33 fragment successively from C, reaction terminates rear methyl alcohol and shrinks; resin vacuum dried overnight, weighs and obtains for degree Shandong peptide (4-33)-PAM resin 74.3g.
Embodiment 15:H-Gly-Ser, (tBu)-Phe-Ser, (tBu)-Asp, (OtBu)-Glu, (OtBu)-Met-Asn, (Trt)-Thr, (tBu)-Ile-Leu-Asp, (OtBu)-Asn, (Trt)-Leu-Ala-Ala-Arg, (pbf)-Asp, (OtBu)-Phe-Ile-Asn, (Trt)-Trp, (Boc)-Leu-Ile-Gln, (Trt)-Thr-Lys, (Boc)-Ile-Thr, (tBu)-Asp, (OtBu)--the preparation of HMPA-AM Resin peptide resin
Take Fmoc-Asp (the OtBu)-HMPA-AM resin 32.4g that substitution degree in embodiment 10 is 0.309mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Get 11.93g Fmoc-Thr (tBu)-OH(30mmol), 4.86g HOBt(36mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, under ice-water bath, add 5.63mLDIC(36mmol) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to replacing degree Shandong peptide main chain peptide sequence; N is held to hold the coupling completing 4-33 fragment successively from C; reaction terminates rear methyl alcohol and shrinks; resin vacuum dried overnight, weighs and obtains for degree Shandong peptide (4-33)-HMPA-AM resin 75.2g.
Embodiment 16:H-Gly-Ser, (tBu)-Phe-Ser, (tBu)-Asp, (OtBu)-Glu, (OtBu)-Met-Asn, (Trt)-Thr, (tBu)-Ile-Leu-Asp, (OtBu)-Asn, (Trt)-Leu-Ala-Ala-Arg, (pbf)-Asp, (OtBu)-Phe-Ile-Asn, (Trt)-Trp, (Boc)-Leu-Ile-Gln, (Trt)-Thr-Lys, (Boc)-Ile-Thr, (tBu)-Asp, (OtBu)--the preparation of HMPA-MBHA Resin peptide resin
Take Fmoc-Asp (the OtBu)-HMPA-MBHA resin 32.3g that substitution degree in embodiment 13 is 0.310mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Get 11.93g Fmoc-Thr (tBu)-OH(30mmol), 4.86g HOBt(36mmol), be dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1, under ice-water bath, add 5.63mL DIC(36mmol) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to replacing degree Shandong peptide main chain peptide sequence; N is held to hold the coupling completing 4-33 fragment successively from C; reaction terminates rear methyl alcohol and shrinks; resin vacuum dried overnight, weighs and obtains for degree Shandong peptide (4-33)-HMPA-MBHA resin 75.5g.
The synthesis of embodiment 17:Fmoc-Asp (OtBu)-CTC resin
Take the CTC resin 100g that substitution degree is 1.0mmol/g, join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Take 82.3gFmoc-Asp (OtBu)-OH(200mmol) be dissolved in DMF solution, add 69.5mLDIPEA(400mmol under ice-water bath) activate 3min after add in solid state reaction post, again add 34.8mLDIPEA(200mmol after room temperature reaction 5min).Room temperature reaction 60min.Wash 3 times with DMF, add 81mL confining liquid (methyl alcohol: 2000mmol) and close 8 hours (if resin does not spread completely add DMF as solvent).Wash 6 times with DMF, methyl alcohol shrinks to be drained, and obtains Fmoc-Asp (OtBu)-CTC resin.Detection substitution degree is 0.916mmol/g.
The preparation of embodiment 18:Boc-His (Trt)-Gly-Asp (OtBu)-CTC peptide resin
Take Fmoc-Asp (the OtBu)-CTC resin 98.3g(90mmol that substitution degree in embodiment 17 is 0.916mmol/g), join in solid state reaction post, wash 2 times with DMF, with DMF swellable resins 30 minutes.Get 80.271gFmoc-Gly-OH(270mmol), 43.8g HOBt(324mmol), be dissolved in DMF solution, under ice-water bath, add 50.7mL DIC(324mmol) activate 3min after add in solid state reaction post, room temperature reaction 2 hours.Detect with ninhydrin method and judge reaction end, if resin water white transparency, then represent and react completely; Resin develops the color, then represent that reaction not exclusively, needs linked reaction 1 hour again, and this judging criterion is applicable to detect with ninhydrin method in subsequent content judge reaction end.Repeat the step that the above-mentioned Fmoc of removing protects and adds corresponding amino acid couplings; according to replacing degree Shandong peptide main chain peptide sequence; complete the solid phase coupling of Boc-His (Trt)-OH again; reaction terminates rear methyl alcohol and shrinks; resin vacuum dried overnight, weighs and obtains Boc-His (Trt)-Gly-Asp (OtBu)-CTC126.1g.
The preparation of embodiment 19:Boc-His (Trt)-Gly-Asp (OtBu)-OH
Boc-His (Trt)-Gly-Asp (OtBu)-CTC peptide resin embodiment 18 prepared is placed in scission reaction bottle, adds lytic reagent (TFA:DCM=1:99(V/V) with the ratio of 10mL/g resin), stirring at room temperature 2.5h.Reaction solution sand core funnel filters, and collect filtrate, resin washs 3 times with a small amount of DCM again, concentrating under reduced pressure after merging filtrate.Add freezing anhydrous diethyl ether precipitation, collecting precipitation anhydrous diethyl ether washs 3 times, and vacuum-drying obtains white powder solid, i.e. thick peptide Boc-His (Trt)-Gly-Asp (OtBu)-OH.Weight 63.2g, HPLC purity is 96.1%.
Embodiment 20: for the synthesis of degree Shandong peptide peptide resin
Take the 76.0g(10mmol prepared by the method for embodiment 4) the preparation of H-Gly-Ser (tBu)-Phe-Ser (tBu)-Asp (OtBu)-Glu (OtBu)-Met-Asn (Trt)-Thr (tBu)-Ile-Leu-Asp (OtBu)-Asn (Trt)-Leu-Ala-Ala-Arg (pbf)-Asp (OtBu)-Phe-Ile-Asn (Trt)-Trp (Boc)-Leu-Ile-Gln (Trt)-Thr-Lys (Boc)-Ile-Thr (tBu)-Asp (OtBu)-Wang Resin peptide resin, add in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins 30 minutes.Boc-His (Trt)-Gly-Asp (OtBu)-OH crude product (30mmol) prepared by 21.7g embodiment 19,11.41g HATU(30mmol), 4.90g HOAt(36mmol) be dissolved in DMF, 10.43mL DIPEA is dripped in ice bath, add in solid state reaction post after activation 5min, room temperature reaction 2h.Reaction end detects with ninhydrin method and is as the criterion, if resin water white transparency, then reacts completely, and resin develops the color, and represents that reaction not exclusively, needs linked reaction 1h again.Reaction terminates rear methyl alcohol and shrinks, peptide resin dried in vacuo overnight, the Boc-His that weighs (Trt)-Gly-Asp (OtBu)-(Hmb) Gly-Ser (tBu)-Phe-Ser (tBu)-Asp (OtBu)-Glu (OtBu)-Met-Asn (Trt)-Thr (tBu)-Ile-Leu-Asp (OtBu)-Asn (Trt)-Leu-Ala-Arg (pbf)-Asp (OtBu)-Phe-Ile-Asn (Trt)-Trp (Boc)-Leu-Ile-Gln (Trt)-Thr-Lys (Boc)-Ile-Thr (tBu)-Asp (OtBu)-Wang Resin peptide resin is 83.2g.
Embodiment 21: for the synthesis of degree Shandong peptide peptide resin
Take the 74.3g(10mmol prepared by the method for embodiment 14) the preparation of H-Gly-Ser (tBu)-Phe-Ser (tBu)-Asp (OtBu)-Glu (OtBu)-Met-Asn (Trt)-Thr (tBu)-Ile-Leu-Asp (OtBu)-Asn (Trt)-Leu-Ala-Ala-Arg (pbf)-Asp (OtBu)-Phe-Ile-Asn (Trt)-Trp (Boc)-Leu-Ile-Gln (Trt)-Thr-Lys (Boc)-Ile-Thr (tBu)-Asp (OtBu)-Pam Resin peptide resin, add in solid state reaction post, 2 times are washed with DMF, with DMF swellable resins 30 minutes.Boc-His (Trt)-Gly-Asp (OtBu)-OH crude product (30mmol) prepared by 21.7g embodiment 19,11.41g HATU(30mmol), 4.90g HOAt(36mmol) be dissolved in DMF, 10.43mL DIPEA is dripped in ice bath, add in solid state reaction post after activation 5min, room temperature reaction 2h.Reaction end detects with ninhydrin method and is as the criterion, if resin water white transparency, then reacts completely, and resin develops the color, and represents that reaction not exclusively, needs linked reaction 1h again.Reaction terminates rear methyl alcohol and shrinks, peptide resin dried in vacuo overnight, the Boc-His that weighs (Trt)-Gly-Asp (OtBu)-(Hmb) Gly-Ser (tBu)-Phe-Ser (tBu)-Asp (OtBu)-Glu (OtBu)-Met-Asn (Trt)-Thr (tBu)-Ile-Leu-Asp (OtBu)-Asn (Trt)-Leu-Ala-Arg (pbf)-Asp (OtBu)-Phe-Ile-Asn (Trt)-Trp (Boc)-Leu-Ile-Gln (Trt)-Thr-Lys (Boc)-Ile-Thr (tBu)-Asp (OtBu)-Wang Resin peptide resin is 81.5g.
Embodiment 22: for the preparation of the thick peptide of degree Shandong peptide
83.2g Boc-His (the Trt)-Gly-Asp (OtBu) embodiment 20 prepared-(Hmb) Gly-Ser (tBu)-Phe-Ser (tBu)-Asp (OtBu)-Glu (OtBu)-Met-Asn (Trt)-Thr (tBu)-Ile-Leu-Asp (OtBu)-Asn (Trt)-Leu-Ala-Arg (pbf)-Asp (OtBu)-Phe-Ile-Asn (Trt)-Trp (Boc)-Leu-Ile-Gln (Trt)-Thr-Lys (Boc)-Ile-Thr (tBu)-Asp (OtBu)-Wang Resin peptide resin is placed in scission reaction wherein, lytic reagent (TFA: thioanisole: EDT:TIS: water=86:5:5:3:1(V/V) is added) with the ratio of 10mL/g resin, stirring at room temperature 2.5h.Reaction solution sand core funnel filters, and collect filtrate, resin washs 3 times with a small amount of TFA again, concentrating under reduced pressure after merging filtrate.Add freezing anhydrous diethyl ether precipitation, collecting precipitation anhydrous diethyl ether washs 3 times, and vacuum-drying obtains white powder solid, namely replaces thick peptide 36.52g, the HPLC purity of degree Shandong peptide to be 63.8%.
Embodiment 23: for the preparation of the thick peptide of degree Shandong peptide
105.7g Boc-His (the Trt)-Gly-Asp (OtBu) embodiment 21 prepared-(Dmb) Gly-Ser (tBu)-Phe-Ser (tBu)-Asp (OtBu)-Glu (OtBu)-Met-Asn (Trt)-Thr (tBu)-Ile-Leu-Asp (OtBu)-Asn (Trt)-Leu-Ala-Arg (pbf)-Asp (OtBu)-Phe-Ile-Asn (Trt)-Trp (Boc)-Leu-Ile-Gln (Trt)-Thr-Lys (Boc)-Ile-Thr (tBu)-Asp (OtBu)-PAM peptide resin is placed in scission reaction wherein, lytic reagent (TFA: thioanisole: EDT:TIS: water=86:5:5:3:1(V/V) is added) with the ratio of 10mL/g resin, stirring at room temperature 2.5h.Reaction solution sand core funnel filters, and collect filtrate, resin washs 3 times with a small amount of TFA again, concentrating under reduced pressure after merging filtrate.Add freezing anhydrous diethyl ether precipitation, collecting precipitation anhydrous diethyl ether washs 3 times, and vacuum-drying obtains white powder solid, namely replaces thick peptide 34.21g, the HPLC purity of degree Shandong peptide to be 59.78%.
Embodiment 24: for the preparation of degree Shandong peptide essence peptide acetate
To take in embodiment 22 ~ 23 any 30.0g for after degree Shandong peptide thick peptide 3000mL water dissolution, adopt Waters2545RP-HPLC system, wavelength 230nm, chromatographic column is the anti-phase C18 post of 50 × 250mm, conventional 0.2%TFA/ acetonitrile mobile phase purifying, collect object peak cut, obtain purity and be greater than 98.5% smart peptide.Smart peptide solution is adopted Waters2545RP-HPLC system, chromatographic column is the anti-phase C18 post of 50 × 250mm, 0.1% acetum/acetonitrile mobile phase turns salt, collect object peak cut, rotary evaporation concentrates, freeze-drying obtains for degree Shandong peptide acetate essence peptide >=9.0g, RP-HPLC purity >=98.5%, Asp degradation impurity <0.2%.
The explanation of above embodiment just understands method of the present invention and core concept thereof for helping.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also fall in the protection domain of the claims in the present invention.
Claims (10)
1., for a preparation method for degree Shandong peptide, it is characterized in that, comprising:
Step 1: by Fmoc-Asp (OtBu)-OH and solid phase carrier coupling, obtain Fmoc-Asp (OtBu)-Resin;
Step 2:Fmoc-Asp (OtBu)-Resin is according to obtaining for degree Shandong peptide 4-33 fragment peptide resin H-Gly-Ser (tBu)-Phe-Ser (tBu)-Asp (OtBu)-Glu (OtBu)-Met-Asn (Trt)-Thr (tBu)-Ile-Leu-Asp (OtBu)-Asn (Trt)-Leu-Ala-Ala-Arg (pbf)-Asp (OtBu)-Phe-Ile-Asn (Trt)-Trp (Boc)-Leu-Ile-Gln (Trt)-Thr-Lys (Boc)-Ile-Thr (tBu)-Asp (OtBu)-Resin for the degree Shandong peptide order 4-32 amino acids for degree Shandong peptide that coupling Fmoc protects one by one;
Step 3: solid phase synthesis is for degree Shandong peptide 1-3 fragment peptide resin Boc-His (Trt)-Gly-Asp (OtBu)-Resin, and then cracking obtains for degree Shandong peptide full guard 1-3 peptide fragment Boc-His (Trt)-Gly-Asp (OtBu)-OH;
Step 4: for degree Shandong peptide full guard 1-3 peptide fragment, obtain for degree Shandong peptide resin for the coupling in solid phase of degree Shandong peptide 4-33 fragment peptide resin;
Step 5: obtain for the thick peptide of degree Shandong peptide for degree Shandong peptide resin cracking;
Step 6: obtain for degree Shandong peptide sterling for the thick peptide purification of degree Shandong peptide.
2. preparation method according to claim 1, it is characterized in that, solid phase carrier described in step 1) is selected from Wang Resin, PAM Resin, HMPA-AM Resin, HMPA-MBHA Resin resin.
3. preparation method according to claim 1, it is characterized in that, the initial substitution degree of solid phase carrier described in step 1) is 0.5mmol/g ~ 1.0mmol/g.
4. preparation method according to claim 1, is characterized in that, step 2) substitution degree of described Fmoc-Asp (OtBu)-Resin is 0.2mmol/g ~ 0.5mmol/g.
5. preparation method according to claim 1, is characterized in that, step 2) coupling reagent of described coupling is HOBt/DIC.
6. preparation method according to claim 1, it is characterized in that, the carrier of solid phase synthesis described in step 3) is CTC resin.
7. preparation method according to claim 1, it is characterized in that, the cracking agent of cracking described in step 3) is the mixture of TFA and DCM or the mixture of TFE and DCM.
8. preparation method according to claim 1, it is characterized in that, the coupling reagent of coupling described in step 4) is HATU/HOAt/DIPEA.
9. preparation method according to claim 1, it is characterized in that, the cracking agent of cracking described in step 5) is the mixing solutions of TFA, PhSMe, EDT, TIS and water.
10. preparation method according to claim 1, is characterized in that, step 6) described purifying is RPLC purifying.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310369843.4A CN104418949A (en) | 2013-08-22 | 2013-08-22 | Preparation method of teduglutide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310369843.4A CN104418949A (en) | 2013-08-22 | 2013-08-22 | Preparation method of teduglutide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104418949A true CN104418949A (en) | 2015-03-18 |
Family
ID=52969137
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310369843.4A Pending CN104418949A (en) | 2013-08-22 | 2013-08-22 | Preparation method of teduglutide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104418949A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109456404A (en) * | 2018-12-31 | 2019-03-12 | 江苏诺泰澳赛诺生物制药股份有限公司 | A kind of synthetic method for degree Shandong peptide |
| WO2019069274A1 (en) | 2017-10-04 | 2019-04-11 | Chemical & Biopharmaceutical Laboratories Of Patras S.A. | A process for preparing a glucagon-like peptide |
| CN111018962A (en) * | 2019-12-27 | 2020-04-17 | 中肽生化有限公司 | Method for preparing teduglutide based on solid phase step-by-step method |
| CN111944037A (en) * | 2019-04-30 | 2020-11-17 | 深圳市健元医药科技有限公司 | Synthetic method of somaglutide |
| CN112175067A (en) * | 2019-05-28 | 2021-01-05 | 深圳市星银医药有限公司 | Preparation method of teduglutide |
| CN113943362A (en) * | 2020-07-15 | 2022-01-18 | 南京星银药业集团有限公司 | Purification method of teduglutide dimer |
| WO2022144860A1 (en) * | 2021-01-04 | 2022-07-07 | Biocon Limited | Synthesis of teduglutide |
-
2013
- 2013-08-22 CN CN201310369843.4A patent/CN104418949A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| CEBRIAN,J ET AL.: "Synthesis of peptide sequences related to thrombospondin: factors affecting aspartimide by-product formation", 《JOURNAL OF PEPTIDE RESEARCH》 * |
| MERGLER,M ET AL.: "The Aspartimide Problem in Fmoc-based SPPS. Part I", 《JOURNAL OF PEPTIDE SCIENCE》 * |
| 康建磊等: "关于合成多肽药物中非对映异构体杂质的研究", 《中国现代应用药学》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019069274A1 (en) | 2017-10-04 | 2019-04-11 | Chemical & Biopharmaceutical Laboratories Of Patras S.A. | A process for preparing a glucagon-like peptide |
| CN109456404A (en) * | 2018-12-31 | 2019-03-12 | 江苏诺泰澳赛诺生物制药股份有限公司 | A kind of synthetic method for degree Shandong peptide |
| CN111944037A (en) * | 2019-04-30 | 2020-11-17 | 深圳市健元医药科技有限公司 | Synthetic method of somaglutide |
| CN111944037B (en) * | 2019-04-30 | 2023-06-13 | 深圳市健元医药科技有限公司 | Synthetic method of somalupeptide |
| CN112175067A (en) * | 2019-05-28 | 2021-01-05 | 深圳市星银医药有限公司 | Preparation method of teduglutide |
| CN112175067B (en) * | 2019-05-28 | 2022-04-15 | 深圳市星银医药有限公司 | Preparation method of teduglutide |
| CN111018962A (en) * | 2019-12-27 | 2020-04-17 | 中肽生化有限公司 | Method for preparing teduglutide based on solid phase step-by-step method |
| CN113943362A (en) * | 2020-07-15 | 2022-01-18 | 南京星银药业集团有限公司 | Purification method of teduglutide dimer |
| WO2022144860A1 (en) * | 2021-01-04 | 2022-07-07 | Biocon Limited | Synthesis of teduglutide |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104418949A (en) | Preparation method of teduglutide | |
| CN106699871B (en) | Preparation method of liraglutide | |
| WO2018032521A1 (en) | Method for synthesizing liraglutide | |
| CN102286092B (en) | Solid-phase synthesis method of liraglutide | |
| JP2019503369A (en) | Method for producing semaglutide | |
| KR101297942B1 (en) | Synthesis of glucagon-like peptide | |
| CN103848910B (en) | The solid phase synthesis process of a kind of Sa Molutai | |
| CN110372785B (en) | Synthesis method of Somalutide | |
| CN112010961B (en) | Solid-liquid synthesis method of somalupeptide | |
| CN101357938B (en) | Method for synthesizing Exenatide from solid phase polypeptide | |
| CN103087181A (en) | Solid-phase synthesis method of liraglutide | |
| CN104072605B (en) | A kind of preparation method for degree Shandong peptide | |
| CN101463081A (en) | GLP-1 derivative | |
| CN103467575B (en) | A kind of preparation method of Pa Xirui peptide | |
| CN111087462B (en) | Solid-phase synthesis method of abamectin | |
| WO2020199461A1 (en) | Method for synthesizing polypeptide-derived compound | |
| CN112679602A (en) | Solid-phase synthesis method of Somaloutide | |
| CN104817638B (en) | A method of synthesis is for degree Shandong peptide | |
| CN102532303A (en) | Synthesis and application of exenatide joined with polyethylene glycol | |
| EP2915817B1 (en) | Method for preparing exenatide | |
| CN103641894A (en) | Preparation method of polypeptide medicine for treating cushing disease | |
| CN107778351A (en) | A kind of method of full synthesis in solid state Octreotide | |
| CN104817631A (en) | Method for synthesizing sinapultide | |
| WO2023279323A1 (en) | Method for synthesizing glp-1 analog | |
| CN110330560B (en) | Synthetic method of lanreotide acetate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150318 |
|
| RJ01 | Rejection of invention patent application after publication |